Organism reproduction, development, and survival are dependent on the equal and accurate segregation of genetic material into two daughter cells. The cell ensures the fate of DNA by properly assembling a macromolecular structure called the mitotic spindle to align and segregate chromosomes. The spindle is composed of microtubules (MTs) and many associated proteins that regulate MT dynamics to organize and shape the spindle. There are many proposed models for how the spindle assembles, however, the contributions of centrosomes, kinetochores, and chromatin to this process are not clearly defined. Although centrosomes are a primary source of MT nucleation in somatic cells, chromatin-mediated MT nucleation also occurs. Chromatin-mediated spindle assembly is mediated by the RanGTP and Chromosome Passenger Complex (CPC) gradients in which downstream proteins of these gradients regulate MT dynamics and are important for spindle organization. Many cell cycle related therapeutic techniques are used to alter MT dynamics or motor proteins that are involved in spindle assembly. Therefore, understanding how the spindle is initially organized will be beneficial in characterizing the activity of MT associated proteins that are the targets of therapeutic agents. In this present proposal I will: 1) Determine how the spindle is organized in the absence of chromatin and kinetochores in which I will determine how MTs are nucleated and organized in spindles formed in the absence of these components in two model systems. 2) Define the mechanisms utilized by the RanGTP and CPC gradients for spindle assembly to test the hypothesis that in a normal cell these two gradients overlap and cooperate for proper spindle formation. I will test this model using a FRET-based sensor of the CPC to detect the distribution of the gradient when the RanGTP gradient has been suppressed. Together these experiments will allow for further understanding of how these MT nucleating factors coordinate for proper spindle organization.